I0634

The conditionally immortalized mouse podocyte cell line was kindly donated by Professor Shengqiang Yu from Shanghai Changzheng Hospital. Briefly, podocytes were cultured as previously shown.²¹ (link) Podocytes were starved overnight before experiments, and treated with LPS (20 μg/ml, L4391, Sigma), CsA (5 μg/ml, C1832, Sigma), FK506 (10 μg/ml, F4679, Sigma), TAK-242(1 μM, HY-11109, MCE), Ionomycin (0.5 μM, I0634, Sigma) or PDTC (20 μM, S1808, Beyotime) for indicated time. Mouse Glomerular Endothelial cells (mGEnC) was provided by Professor Chuanming Hao at Ruijin Hospital, and were cultured in RPMI 1640 medium with 10% FBS in a humidified atmosphere of 5% CO₂. Human Mesangial Cells (HMC) was bought from ATCC, and was cultured in DMEM medium with 10% FBS in a humidified atmosphere of 5% CO₂.

Human HT-1080 fibrosarcoma, A431 epidermoid carcinoma, MCF-7 breast cancer, and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/liter), l-glutamine, and sodium pyruvate (11995073, Gibco). Medium was supplemented with 10% heat-inactivated FBS (16140071, Gibco) and 1% penicillin-streptomycin (10,000 U/ml; 15140122, Gibco). Cells were maintained at 37°C with 5% CO₂. A431 cells expressing GFP-E-cad were a gift from V. Bruntons (EH4 2XR, University of Edinburgh, UK) (63 (link)). In select experiments, cells were treated with the following pharmacological agents: Y-27632 (20 μM; Y0503, Sigma-Aldrich), ionomycin (0.5 and 1 μM; I0634, Sigma-Aldrich), blebbistatin (50 μM; B0560, Sigma-Aldrich), Rho inhibitor I (1 μg/ml; CT04-A, Cytoskeleton Inc.), LPA (50 μM; L7260, Sigma-Aldrich), paclitaxel (taxol equivalent) (1 μM; P3456, Thermo Fisher Scientific), colchicine (125 μM; C9754, Sigma-Aldrich), and importazole (50 μM; SML0341, Sigma-Aldrich).

For ionomycin/histamine treatments, 5 × 10⁵ cells were seeded in 35 mm dishes. The following day, regular growth medium was replaced by 1 ml of live-cell medium (DMEM with 25 mM d-glucose, 4 mM d-glutamine, and 25 mM HEPES, supplemented with 10% newborn calf serum). Another 1 ml of live-cell medium containing 8 µM ionomycin (I0634; Sigma; from a 1 mM stock in DMSO) was added swiftly in a circular motion onto the plate, making the final ionomycin concentration 4 µM. The same procedure was applied for histamine treatments, with a final concentration of 100 µM (H7125; Sigma; from a 100 mM stock in DMSO). Stimulations were carried out for the indicated time durations (Figure 1, B and E), followed by instant medium removal and cell lysis for Western blotting.